Development 142: doi:10.1242/dev.119008: Supplementary Material
Figure S1: yap-/- mutants exhibit RPE defects noticeable at the onset of
pigmentation.
(A-A’’) A Wild-type embryo with normal RPE cells and pigmentation encompassing the
whole eye globe.
(B-B’’) yap-/- embryos lack RPE cells before RPE cells are completely pigmented.
White arrows = areas of absent of RPE cells.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Figure S2: Overexpression of NLS-YapDN and NLS-TazDN in rx3 positive cells
results in loss of RPE.
(A-C) Examples of eyes from rx3:gal4+/dsRed:uas:NLS-YapDN and
rx3:gal4+/dsRed:uas:NLS-TazDN fry lacking RPE cells at 48 hpf.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Figure S3: qRT-PCR validates mRNA transcripts that were upregulated via
RNAseq in Yap S87A overexpressing 36 hpf eyes and -1.0kb ctgfa:d2GFP is
upregulated by Yap S87A.
(A) ctgfa (9.5-fold, p=0.0508), cyr61 (12.6-fold, p=0.1072), clu (135.5-fold, p=0.1705),
fn1b (195-fold, p=0.2418), hbegfa (2.4 –fold, p=0.0845), lats2 (6.7-fold, p=0.0005),
pawrl (17.6-fold, p=0.1736). Dashed line indicates the normalized expression levels of
yap and taz in wild-type embryos. An unpaired t-test was performed on mRNA
expression and statistical significance was performed using the Holm-Sidak method.
Error bars = s.e.m. (B-B’) -1.0kb ctgfa:d2GFP expression is observed in the NR, RPE,
heart, and other tissues at 28 hpf. (C-F’’) Yap S87A overexpression results in increased
-1.0kb ctgfa:d2GFP in RPE cells (F-F’’) and ectopic NR expression (E-E’’) compared to
mCherry (C-D’’). Arrows=ectopic NR, arrow heads=RPE expression. All embryos are
28 hpf.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Figure S4: Overexpression of Yap S87A in vsx2 positive cells results in ectopic
expression of dct mRNA and -2.7kb tfec:Gal4-VP16/dsRed:UAS:Yap S87A positive
cells do not migrate into the neural retina.
(A) Endogenous dct expression is observed in the RPE.
(B-B’’) Ectopic dct expression is observed in the neural retina and enhanced in the
ciliary marginal zone in Yap S87A overexpressing embryos. Black arrows denote
ectopic dct.
(C-C’’’) RPE cells overexpressing Yap S87A do not ectopically migrate into the neural
retina. Images represent a 2 hour time course from 40-42 hpf. White arrows indicate a
Yap S87A positive RPE cell.
Development | Supplementary Material
Development 142: doi:10.1242/dev.119008: Supplementary Material
Movie 1: Time lapse of zebrafish eye morphogenesis including various transcription
factors and signaling pathways involved.
Movie 2: 4xGTIIC:d2GFP transgene expression during optic cup morphogenesis.
Arrows indicate RPE and lens expression. L=lens, NR=neural retina, RPE=retina
pigment epithelium.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Movie 3: -2.7kb tfec:eGFP/h2afz:H2A-mCherry transgene expression from 14-24
hpf during optic cup morphogenesis.
Movie 4: -2.7kb tfec:eGFP/h2afz:H2A-mCherry expression from 14-24 hpf during
optic cup morphogenesis. The arrow represents a -2.7kb tfec:eGFP+ mitotic cell.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Movie 5: -2.7kb tfec:Gal4-VP16/dsRed:UAS:Yap S87A/H2A-eGFP expression from
14-25 hpf during optic cup morphogenesis. The arrows highlight -2.7kb tfec:Gal4VP16/dsRed:UAS:Yap S87A positive cells that migrating normally around the rim of the
optic cup and not into the presumptive neural retina. No abnormal RPE cell migrations
were noted. The time lapse movie is played twice.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Movie 6: -2.7kb tfec:Gal4-VP16/dsRed:UAS:Yap S87A/H2A-eGFP expression from
36-48 hpf during retinal neurogenesis. The arrows highlight -2.7kb tfec:Gal4VP16/dsRed:UAS:Yap S87A positive cells that maintain their position in the RPE and do
not migrate into the neural retina. No abnormal RPE cell migration was noted. The time
lapse movie is played twice.
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Table S1: The loss of RPE phenotype observed in yap-/- embryos is rescued when
embryos are reared at 20.5°C. Embryos were placed at 20.5°C at 70% epiboly and put
back at 28.5°C at prim-10.
yap+/- X yap+/28.5°C
Total Embryos Scored
Wild Type RPE
Mutant RPE
% of Mutant/Wild Type RPE
Predicted
Values
389
292
97
25.00%
Actual
Values
389
307
82
21%
20.5°C
Total Embryos Scored
Wild Type RPE
Mutant RPE
% of Mutant/Wild Type RPE
Predicted
Values
389
292
97
25%
Actual
Values
389
389
0
0%
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Table S2: The loss of RPE phenotype cannot be completely rescued by 20.5°C
rearing when a mutant taz allele is present in the yap-/- background. Embryos were
placed at 20.5°C at 70% epiboly and put back at 28.5°C at prim-10.
yap+/-;taz+/- X yap-/-;taz+/+
28.5°C
Total Embryos Scored
Wild Type RPE
Mutant RPE
% of Mutant/Wild Type RPE
Predicted
Values
136
68
68
50%
Actual
Values
136
74
62
46%
20.5°C
Total Embryos Scored
Wild Type RPE
Mutant RPE
% of Mutant/Wild Type RPE
Predicted
Values
135
68
67
50%
Actual
Values
135
110
25
19%
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Development 142: doi:10.1242/dev.119008: Supplementary Material
Table S3: The top 20 most upregulated transcripts in Yap S87A overexpressing 36
hpf eyes. Values represent the fold change of Yap S87A expressing eyes compared to
sibling controls. All transcripts were significantly upregulated based on an adjusted pvalue < 0.05.
Transcript ID
Gene
Fold
Change
ENSDART00000060765
BX323876.3
187.12
ENSDART00000141193
clu
179.31
ENSDART00000109972
BX248501.1
91.70
ENSDART00000129496
cyr61
33.07
ENSDART00000018117
ppp1r14aa
20.69
ENSDART00000037904
socs3b
19.77
ENSDART00000109138
hbegfa
19.59
ENSDART00000104965
plcxd1 (3 of 5)
18.04
ENSDART00000003505
adm (1 of 2)
17.12
ENSDART00000112226
apcdd1l
16.35
ENSDART00000077951
pcolce2b
15.92
ENSDART00000017312
fn1b
15.27
ENSDART00000097460
hmgcra
15.23
ENSDART00000148845
DKEY-6N3.3
12.97
ENSDART00000063028
ctgf
12.50
ENSDART00000112243
crlf1a
11.36
ENSDART00000124465
junbl
10.79
ENSDART00000150088
DKEY-119G10.4
10.53
ENSDART00000106488
plod2
10.18
ENSDART00000145103
cntfr
9.91
Development | Supplementary Material
Development 142: doi:10.1242/dev.119008: Supplementary Material
Table S4: List of primers used for genotyping and qRT-PCR. E=Efficiency % of
qRT-PCR primers.
Primer Name
yap 4bp/21bp F
yap 4bp/21bp R
taz 5bp F
taz 5bp R
yap qPCR F
Primer Sequence
5’-AGTCATGGATCCGAACCAGCACAA-3’
5’-TGCAATCGGCCTTTATTTTCCTGC-3’
5’-CTCGGCTGAAACTACTTAAGGACG-3’
5’-CTAAACAGTGTGCAGGAATGTCC-3’
5’-CCAGACAAGCCAGTACAGAT-3’
yap qPCR R
5’-GAAGTATCTCTGTCCCGAAGG-3’
taz qPCR F
5’-GCATCCAGATGGAGAGAGAG-3’
taz qPCR R
5’-GCTGTTATTGGGCATGTTTC-3’
ef1α exon1-2 F
5’-TCTCTCAATCTTGAAACTTATCAATCA-3’
ef1α exon3 R
5’-AACACCCAGGCGTACTTGAA-3’
ctgfa F
5’-CTGCACAGCCAGAGATG-3’
ctgfa R
5’-CACTTCCCAGGCACTTT-3’
cyr61 F
5’-CCGTGTCCACATGTACATGGG-3’
cyr61 R
5’-GGTGCATGAAAGAAGCTCGTC-3’
clu F
5’-GTCGCAAGTTGGTGAGAAATACC-3’
clu R
5’-CTCCTTCATCTCCTGAGCCATC-3’
fn1b F
5’-CAGTACTGTACAGTCAGGGGAAGC-3’
fn1b R
5’-CACGACCGTTGTCATTACAGCC-3’
hbegfa F
5’-CAAGCAAGGTGCATATAATGTGG-3’
hbegfa R
5’-CTGCCAAACAAACACGGTCAC-3’
Development | Supplementary Material
Assay
genotyping
genotyping
genotyping
genotyping
RT-qPCR
E=95%, r2=.997
RT-qPCR
E=95%, r2=.997
RT-qPCR
E=105%, r2=.991
RT-qPCR
E=105%, r2=.991
RT-qPCR
E=102.7%,
r2=.992
RT-qPCR
E=102.7%,
r2=.992
RT-qPCR
E=120%, r2=.990
RT-qPCR
E=120%, r2=.990
RT-qPCR
E=107.7%,
r2=.987
RT-qPCR
E=107.7%,
r2=.987
RT-qPCR
E=98.7%, r2=.900
RT-qPCR
E=98.7%, r2=.900
RT-qPCR
E=94.7%, r2=.970
RT-qPCR
E=94.7%, r2=.970
RT-qPCR
E=105.6%,
r2=.980
RT-qPCR
E=105.6%,
Development 142: doi:10.1242/dev.119008: Supplementary Material
lats2 F
5’-CTCCGAGAGATCCGCAAGTC-3’
lats2 R
5’-CACGTACAATCTGTTCAGTGTG-3’
pawrl F
5’-GAACAAGACCTTGCTGAAAGTG-3’
pawrl R
5’-CACTTCCACAATCCAAAGCGTCC-3’
Development | Supplementary Material
r2=.980
RT-qPCR
E=93%, r2=.974
RT-qPCR
E=93%, r2=.974
RT-qPCR
E=104%, r2=.906
RT-qPCR
E=104%, r2=.906
Development 142: doi:10.1242/dev.119008: Supplementary Material
Table S5. Transgenic and mutant zebrafish lines
Line
Reference
Tg(4xGTIIC:d2GFP)mw50
Miesfeld and Link, 2014
Tg(dsRed.T4:14xUAS:Yap)mw63
Miesfeld and Link, 2014
Tg(dsRed.T4:14xUAS:Wwtr1)mw64
Miesfeld and Link, 2014
Tg(dsRed.T4:14xUAS:YapCA(S87A))mw65
Miesfeld and Link, 2014
Tg(dsRed.T4:14xUAS:NLS-YapDN)mw66
This study
Tg(dsRed.T4:14xUAS:NLS-TazDN)mw67
This study
Tg(2.7 kb tfec:eGFP)mw68
This study
Tg(dsRed.T4:14xUAS:Myc-TeadY417H)mw70
This study
Tg(2.7 kb tfec:Gal4-VP16) mw71
This study
Tg(5.0 kb foxC1b:Gal4-VP16)mw72
This study
Tg (1.0 kb ctgfa:d2GFP)mw75
This study
Tg(vsx2:Gal4-VP16)mw39
Clark et al., 2011
yap c.158_161delmw48
This study
yap c.158_178delmw69
This study
wwtr1 c.156_160delmw49
taz mutants (this study)
yapnl13
This study
Tg(h2afv:h2afv-GFP)kca6
Pauls et al., 2001
Tg(Ola.Rx3:Gal4-VP16)vu271
Weiss et al., 2012
albinob4, slc45a2b4/b4
Streisinger et al., 1986, Tsetskhladze et al., 2012
Development | Supplementary Material
Development 142: doi:10.1242/dev.119008: Supplementary Material
Supplementary references
Pauls, S., Geldmacher-Voss, B. and Campos-Ortega, J. A. (2001). A zebrafish histone variant H2A.F/Z
and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development. Dev. Genes
Evol. 211, 603-610.
Weiss, O., Kaufman, R., Michaeli, N. and Inbal, A. (2012). Abnormal vasculature interferes with optic
fissure closure in lmo2 mutant zebrafish embryos. Dev. Biol. 369, 191-198.
Development | Supplementary Material